A fundamental problem faced by all cells is the need to translocate proteins across and into cellular membranes. It is now clear that some distantly-related membranes employ translocation mechanisms that are evolutionarily related while other membranes employ apparently unique mechanisms. The goal of this project is to elucidate mechanisms by which proteins are targeted to the chloroplast thylakoid membrane, a membrane that plays a central role in providing the fixed carbon that supports terrestrial life. Enormous progress has been made in understanding this process recently, due largely to the application of in vitro targeting assays. These experiments provided evidence for several energetically- distinct pathways by which proteins can translocate into or across the thylakoid. Two of these pathways are related to previously described mechanisms while a third appears novel. To fully appreciate the relationships between targeting pathways, the nature of the targeting machineries, and the roles in vivo of factors identified in biochemical studies, it is essential to study the consequences of genetic disruption of thylakoid protein targeting. We have developed a genetic system in maize with which we can analyze this process. Previously, we isolated mutations that disrupt thylakoid protein targeting and cloned two of the corresponding genes. The experiments proposed more fully exploit the genetics of this system to address the following questions: a) What are the components of the machinery that translocates proteins across the thylakoid? This question will be addressed by cloning two new transposon-tagged targeting mutations, by identifying proteins that interact with the products of these genes, and by identifying additional targeting mutations. b) What roles do the thylakoid translocation machineries play in the integration of thylakoid proteins? This question will be addressed by analyzing the rates of integration of such proteins in mutants in which translocation across the membrane is disrupted. c) How do the different thylakoid targeting pathways interact in vivo? To establish redundancies and compensatory interactions between pathways, targeting defects in double mutants and the accumulation of targeting machinery components in single mutants will be analyzed. Prior evidence suggests that the ~novel" thylakoid targeting pathway may be related to a universal targeting mechanism that has previously escaped detection. Therefore, the results of these experiments will elucidate not only the essential process of thylakoid membrane assembly but may also provide insight into targeting mechanisms employed by all cells.